In all animals, the germ cells (precursors to eggs and sperm) form very early in development. The region of the embryo destined to make germ cells contains special structures called polar granules in Drosophila. This proposal is aimed toward using Drosophila to increase our understanding of the mechanism of germ cell formation and development. The major focus will be on Vasa (Vas), a protein which is localized in polar granules and essential for the development of germ cells in Drosophila. We have genetic evidence implicating Vas as a germ-line specific translational activator. We plan a series of experiments aimed at identifying target mRNAs for Vas and at understanding how Vas may regulate translation. Genetic evidence indicates that translation of two specific mRNAs, Bic-C and grk, requires vas function. We will use reporter-gene constructs to map the elements in these two RNAs which are essential for translational regulation, and will identify proteins which bind to these elements by UV-crosslinking experiments. Two new Vas- interacting proteins we recovered in a yeast two-hybrid screen will also be characterized. One of these new Vas-interacting proteins (Al) is the Drosophila homologue of the S. cerevisiae Fun12p, which is highly related to prokaryotic and mitochondrial translation initiation factors-2. Evidence from other workers implicates Fun12p in translation initiation at the stage of ternary complex formation. We will biochemically purify A1-and Vas-containing complexes to try to link these proteins with known translation initiation factors. The other interacting protein (A11) co-immunoprecipitates from Drosophila tissue extracts with Vas, and colocalizes with Vas in the pole plasm of early embryos. We will isolate genetic mutations in the gene encoding A11 to understand its developmental function. In the long term, we plan to develop a cell-free system for studying germ-line specific translational regulation in Drosophila. We expect that our work will provide important information on the formation and maturation of germ cells, which will ultimately provide a fundamental framework for understanding the basis of many problems leading to human infertility.